Abhinav Sinha

Introduction: Angiotensin II (Ang II) has been proposed to protect from severe malaria by killing the parasite and maintaining the blood-brain barrier integrity by acting through type 1 (AT ₁ R) and type 2 (AT ₂ R) receptors. However, this protection from severe malaria is counterproductive in adulthood as the elevated plasma Ang II puts the individual at risk for developing hypertension. Various natural and synthetic peptides of Ang II have been explored to check its antiplasmodial actions despite Ang II being a significant vasopressor via its actions through AT ₁ R. Therefore, to be able to explore Ang II as a potential antiplasmodial agent, it is important to demonstrate its IC ₅₀ against P. falciparum in vitro and its minimum effective concentration required for activation of AT ₁ R. It is desirable that IC ₅₀ of Ang II against P. falciparum remains below the threshold for AT ₁ R activation and hence to verify this, the current study was planned as there are no previously published reports for determining the threshold Ang II concentration for AT ₁ R activation. Methods: For invasion inhibition assay, Pf-infected RBCs (schizonts) were incubated with four different concentrations of Ang II for 16 hours. The activation of AT ₁ R and AT ₂ R in the presence of Ang II (from 10 ^–9 M to 10 ^–7 M) was tested by calcium and cGMP signaling assays, respectively using the human brain microvascular endothelial cell line as an in vitro model. Results: Effective Ang II concentration among all tested concentrations was found to be 10 ^–6 M against Plasmodium falciparum invasion. Statistically non-significant and negligible responses in AT ₁ R and AT ₂ R activation were observed below the desired Ang II concentration of 10 ^–6 M. Discussion: The results indicate that the IC ₅₀ of Ang II against P. falciparum is below the threshold for AT ₁ R activation. However, the minimum effective Ang II concentration required for AT ₁ R activation remains unanswered which demands further improvement in experimental designs.

Introduction Malaria continues as a public health threat through symptomatic/febrile cases, asymptomatic and low-density infections of Plasmodium falciparum, P. vivax, and their mixed infections. Mixed infections have not been studied much regarding their burden, clinical manifestations, and implications, and therefore, this study was conducted. Methods Febrile patients were recruited from four patient-care settings from June to November 2020 through the collection of dried blood spots (DBS) and their paired microscopy and/or rapid diagnostic test (RDT) data. Polymerase chain reaction (PCR)-based molecular diagnosis of both parasite species was performed from genomic DNA isolated from the DBS. Clinico-demographic details were recorded from patients from one of the sites, wherein patients with mixed infections were telephonically followed for subsequent clinical development. Results Out of the 1030 samples collected and analyzed, 27% (280) were infected with P. falciparum and/or P. vivax: 188 (18%) mono-P. falciparum, 6 (0.5%) mono-P. vivax and 86 (8%) mixed. None of the infections were detected by microscopy and/or RDT, meaning that all 27% were febrile sub-microscopic infections with 8% burden of mixed infections. The quality of microscopic slides was found to be unsatisfactory when a sub-sample of slides was cross-examined by level 1-competent microscopists. None of the nine mixed-infection patients from Gandhi Medical College and Hospital (GMCH) reported recurrences or any clinical development during the 12-month follow-up. No clinically/statistically significant difference was observed between mono- and mixed infections. Conclusions A high 27% febrile sub-microscopic Plasmodium infections with 8% mixed infections represent a significant challenge for malaria elimination, considering the quality of microscopy and the fact that Madhya Pradesh is classified under category 1 in the National Strategic Plan for malaria elimination 2023–2027.

Introduction: Malaria is still a public health challenge across many regions of the world. With significant efforts, it has become a target for elimination in near future. However, elimination requires early and accurate diagnosis and despite the fact that malaria elimination is nearing in several countries, specific and sensitive diagnosis of malaria and its causative species remains inadequate in many near-elimination settings, including India. With the advent and increasing usage of nucleic acid-based detection of Plasmodium infection, the diagnostic limitations of microscopy, including errors in species identification, have become more prominent. Methods: The purpose of this study was to determine the magnitude of Plasmodium species misidentification by microscopy in India based on previously published reports that performed microscopy and PCR to the same samples. A total of 2706 microscopy-PCR pairs were extracted from 16 different locations across 11 Indian states. Region-specific and species-wise misidentification rates were also estimated. Results: The analyses revealed 15% misidentification rate (408/2706). Surprisingly, microscopy misidentified >98% of mixed-infections (400/405) as mono-infections (almost all as P. falciparum mono-infections). The study identifies Jharkhand and Madhya Pradesh as major contributors (>20%) to Plasmodium species misidentification by microscopy. Discussion: These findings suggest that we are overestimating P. falciparum burden, potentially wasting elimination resources, and underestimating non-falciparum species. The study also addresses an important issue concerning analysis of misidentification and sub-microscopic infection data. It proposes an analysis approach that is expected to help in deciphering misidentification and sub-microscopic infections in a more granular manner, generating actionable data for countries targeting malaria elimination.

India is committed to eliminate malaria by 2030. However, there has been a continuous debate between National Malaria Control Programmes (NMCPs) and World Health rganization (WHO) on the differential perception of malaria burden. The evident gap between the reported cases by NMCPs and WHO estimations, particularly for India, has never been unbiasedly addressed. This work provides a critical understanding of gaps between estimated and reported cases (ERR) of malaria and the possible reasons thereof. We calculated the ratio between estimated and reported cases, called ERR thereof, for highlighting this gap from 2010 to 2022. We also dissected some of the assumptions/adjustments used by WHO to compensate for data inadequacy while estimating malaria cases. The contribution of private sector in reporting malaria cases and contemporary research data on malaria burden in India were correlated with ERR. ERR remained consistently high (WHO-South-East Asia Region >6; India >10) from 2010 to 2022. Certain assumptions/adjustment factors used by WHO for estimation were unclear. Malaria case reporting from private sector in India was virtually non-existent contributing to consistently high ERR. Data from contemporary research in India also reveal a high burden gap with microscopy-positivity being 3 to 55 times higher than that reported by the NMCP. There still remain a few areas where more detailed clarification is needed both from the WHO and the NMCPs reporting malaria burden specifically regarding malaria case reporting from the private health sectors. If these issues are addressed and resolved, the gap between ERR can be narrowed down or even zeroed.

Molecular diagnosis of malaria through nucleic acid-based amplification test is important to detect low-density, sub-microscopic and residual infections, as well as to prevent importations and re-establishment. Reliance on single/limited molecular targets could be detrimental as evidenced by false-negative PfHRP2-based RDTs, and the same may apply to PCR targets. No systematic exploration of the commonly used PCR targets has yet been documented. A systematic search was made using a previously generated database through PubMed® and Google Scholar® and supplemented by additional searches. All studies that used PCR for detecting Plasmodium infections were included in this study. Further information was retrieved on molecular targets used and the type of PCR assay used. An independent search was also made to explore the identification/development of newer molecular targets. Almost all studies (93%) used 18S rRNA gene as a molecular target. Nested PCR alone (68%) was the most frequently used assay. Eighty-five percent of the studies that exploited the 18S rRNA gene target and nested PCR used the approach developed in 1993. Overreliance on a solitary molecular target (18S rRNA gene) for many years might be a cause for concern. Research is needed to validate newer multi-copy targets in terms of limit of detection, robust reproducibility, reduced costs, and a possibility of multiplexing.

Background: India is committed to malaria elimination by the year 2030. According to the classification of malaria endemicity, the National Capital Territory of Delhi falls under category 1, with an annual parasite incidence of Plasmodium vivax cases to the malaria burden in South West Delhi (SWD). Methods: A cross-sectional study was carried out at the fever clinic of the Indian Council of Medical Research-National Institute of Malaria Research in SWD from January 2017 to December 2019. Demographic and travel history data were recorded for all P vivax confirmed malaria cases diagnosed at the fever clinic. Vector and fever surveys along with reactive case detection were conducted in SWD and Bulandshahr district of Uttar Pradesh, 1 of the 6 geographical sources for a high number of imported malaria cases. Results: A total of 355 P vivax malaria cases were reported during the study period. The proportion of imported cases was 63% (n=222). Of these, 96% (n=213) of cases were from Uttar Pradesh. The distribution of malaria cases revealed that imported cases were significantly associated with travel during the transmission season compared with that in the nontransmission season. Entomological and fever surveys and reactive case detection carried out in areas visited by imported P vivax malaria cases showed the presence of adults and larvae of Anopheles species and P vivax parasitemia. Conclusions: Population movement is a key challenge for malaria elimination. Although additional P vivax infections and vector mosquitoes were detected at places visited by the imported malaria cases, the inability to detect the parasite in mosquitoes and the possibility of relapses associated with P vivax limit the significance of malaria associated with the travel. However, there remains a need to address migration malaria to prevent the introduction and re-establishment of malaria in areas with very low or 0 indigenous cases.